Senescence recovering by dual drug-encapsulated liposomal nanoparticles for large-scale human cell expansion.

Although regenerative therapy and bioartificial tissues and organs require a sufficient number of human cells, current cell expansion processes are accompanied by accumulation of senescent cells that are related to deterioration of cellular functions and induction of senescence-associated secretory phenotype (SASP). Therefore, suppression of replicative senescence during expansion is one of the crucial issues for dissemination of regenerative medicine. We herein developed dual drug-encapsulated liposomal nanoparticles (LNPs) to suppress cellular senescence in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and natural killer (NK) cells by removal of dysfunctional mitochondria from the senescent cells. We found that LNP treatment reduced senescent makers; downregulation of p21 expression and reduction of SA-ß-Gal activity in both cells provably due to mitophagy reactivation in the cells. Moreover, SASP secretion in hAT-MSCs and tumor cytotoxicity in NK cells were also improved upon LNP treatments. These findings may contribute to the production of highly effective expanded cells for regenerative medicine and bioartificial tissues and organs.

Gas Adsorption and Diffusion Behaviors in Interfacial Systems Composed of a Polymer of Intrinsic Microporosity and Amorphous Silica: A Molecular Simulation Study.

We investigate the adsorption and diffusion behaviors of CO2, CH4, and N2 in interfacial systems composed of a polymer of intrinsic microporosity (PIM-1) and amorphous silica using grand canonical Monte Carlo (GCMC) and molecular dynamics (MD) simulations. We build model systems of mixed matrix membranes (MMMs) with PIM-1 chains sandwiched between silica surfaces. Gas adsorption analysis using GCMC simulations shows that gas molecules are preferentially adsorbed in microcavities distributed near silica surfaces, resulting in an increase in the solubility coefficients of CO2, CH4, and N2 compared to bulk PIM-1. In contrast, diffusion coefficients obtained from MD simulations and then calibrated using the dual-mode sorption model show different tendencies depending on gas species: CO2 diffusivity decreases in MMMs compared to PIM-1, whereas CH4 and N2 diffusivities increase. These differences are attributed to competing effects of silica surfaces: the emergence of larger pores as a result of chain packing disruption, which enhances gas diffusion, and a quadrupole-dipole interaction between gas molecules and silica surface hydroxyl groups, which retards gas diffusion. The former has a greater impact on CH4 and N2 diffusivities, whereas the latter has a greater impact on CO2 diffusivity due to the strong quadrupole-dipole interaction between CO2 and surface hydroxyls. These findings add to our understanding of gas adsorption and diffusion behaviors in the vicinity of PIM-1/silica interfaces, which are unobtainable in experimental studies.

Fabrication and Electrolyte Characterizations of Nanofiber Framework-Based Polymer Composite Membranes with Continuous Proton Conductive Pathways.

For future fuel cell operations under high temperature and low- or non-humidified conditions, high-performance polymer electrolyte membranes possessing high proton conductivity at low relative humidity as well as suitable gas barrier property and sufficient membrane stability are strongly desired. In this study, novel nanofiber framework (NfF)-based composite membranes composed of phytic acid (Phy)-doped polybenzimidazole nanofibers (PBINf) and Nafion matrix electrolyte were fabricated through the compression process of the nanofibers. The NfF composite membrane prepared from the pressed Phy-PBINf showed higher proton conductivity and lower activation energy than the conventional NfF composite and recast-Nafion membranes, especially at low relative humidity. It is considered that the compression process increased the nanofiber contents in the composite membrane, resulting in the construction of the continuously formed effective proton conductive pathway consisting of the densely accumulated phosphoric acid and sulfonic acid groups at the interface of the nanofibers and the Nafion matrix. Since the NfF also improved the mechanical strength and gas barrier property through the compression process, the NfF composite polymer electrolyte membranes have the potential to be applied to future fuel cells operated under low- or non-humidified conditions.

The potential of lipid-polymer nanoparticles as epigenetic and ROS control approaches for COPD.

Chronic obstructive pulmonary disease (COPD) is a lung disease caused by an inflammatory response to various inhaled toxins, especially cigarette smoke. Reactive oxygen species (ROS) and epigenetic abnormality are intimately related to the pathology of COPD, and the overproduction of ROS results in a decrease of histone deacetylase 2 (HDAC2), leading to glucocorticoid resistance. Therefore, a novel treatment that simultaneously reduces ROS level and glucocorticoid resistance is urgently needed. In this study, we developed a codelivery system using core-shell type lipid-polymer nanoparticles (LPNs) composed of a poly(lactic acid) (PLA) core encapsulating a potent antioxidant Mn-porphyrin dimer (MnPD) and a cationic lipid (DOTAP) shell that binds HDAC2-encoding plasmid DNA (pHDAC2), as a new therapeutic approach toward COPD. The transfection of pHDAC2 combined with the elimination of ROS by MnPD exhibited a significant enhancement of intracellular HDAC2 expression levels, suggesting that the multi-antioxidative activity of MnPD plays a crucial role in the expression of HDAC2. Moreover, treatment with LPNs efficiently ameliorated the steroid resistance in COPD models in vitro as evidenced by the lowered expression levels of IL-8. Recovery from mitochondrial dysfunction may be the mechanism underlying the action of LPNs. The PLA-MnPD/DOTAP/pHDAC2 system proposed offers a new therapeutic approach for COPD based on the synergism of ROS elimination and HDAC2 expression.

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation assay. Furthermore, dynamic light scattering measurement and transmission electron microscopy observations have estimated that the particle size of the resulting MIC was approximately 30 nm, with a rather flexible structure. The APe-Im-E-PEG/pDNA MIC incubated for 2 weeks exhibited hemolytic activity at endosomal pH, presumably because the pH-sensitive carboxyl groups revealed after the hydrolysis of an ester bond of APe-Im-E-PEG. The APe-Im-E-PEG/pDNA MIC enhanced the gene expression 2 weeks after transfection in vivo by intramuscular administration in mice. Consequently, in vivo sustainable gene expression has been achieved by the molecular design of APe-Im-E-PEG for cellular uptake and endosomal escape proceeded by temporal hydrolysis of the ester bond.

New class of artificial enzyme composed of Mn-porphyrin, imidazole, and cucurbit[10]uril toward use as a therapeutic antioxidant.

In this study, we investigated a new class of artificial enzymes composed of Mn-porphyrin, imidazole, and cucurbit[10]uril (CB[10]) toward therapeutic antioxidants. Structural characterization by means of NMR indicated that the inclusion mode of metalloporphyrin in CB[10] was sensitive to the chemical structure of metalloporphyrin and that the structure of the artificial enzyme had a similarly to that of native heme catalase. Kinetic analysis for catalytic antioxidative activities demonstrated that the artificial enzyme exhibited highly efficient activity for H2O2 disproportionation (catalase activity) in water. The activity was classified as top-performing among the water-soluble artificial catalases. The artificial enzyme was constructed by simply mixing the components in water. We consider that this is a great advantage over previously reported artificial catalases, which require a multi-step synthesis or that lack water solubility. The pro-oxidative peroxidase activity was remarkably suppressed due to inclusion in CB[10]. Furthermore, a preliminary in vitro study suggested that the artificial enzyme catalytically eliminated reactive oxygen species, including H2O2, in human cell lines. It was presumed that CB[10] contributed to the bioavailability of the artificial enzyme. Overall, the artificial enzyme was shown to have high potential as a therapeutic antioxidant. We consider that the results in this study could lead to a new conceptual advance toward therapeutic antioxidants that could simultaneously improve the catalytic and biological properties of Mn-porphyrins.

Byproduct-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for in Vivo Protein Delivery.

Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, the addition of poly(ethylene glycol) (PEGylation) methods for the insulin are widely developed. Here, to make these PEGylation methods the simplest, we report the byproduct-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups (that is, Chol-U-Pr-mPEG). The noncovalent PEGylation by the Chol-U-Pr-mPEG has been achieved by the simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution, followed by freeze-drying. The formation of the Chol-U-Pr-mPEG/insulin complex has proceeded without byproducts, such as N-hydroxysuccinimide, formed by the conventional covalent PEGylation using an active ester group. The byproduct-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease. The resulting insulin modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. Consequently, the Chol-U-Pr-mPEG/insulin complex formation offers the byproduct-free intact PEGylation of insulin for in vivo protein delivery.

Structure-activity relationship between Zn2+-chelated alkylated poly(1-vinylimidazole) and gene transfection.

The structure-activity relationship between Zn2+-chelated alkylated poly(1-vinylimidazole) (PVIm) and gene transfection has been demonstrated. From a chemical structure perspective, ethylated PVIm (PVIm-Et) chelated the most Zn2+ ions compared to methylated PVIm (PVIm-Me) and butylated PVIm (PVIm-Bu). The resulting Zn2+-chelated PVIm-Et formed more stable complexes with plasmid DNA complex than non-chelated PVIm-Et. From a biological activity perspective, the Zn2+-chelated PVIm-Et delivered the highest amount of Zn2+ ions inside the cell, corresponding to the highest gene transfection. These results suggest that PVIm-Et is the optimal sequence for the chelation of Zn2+ ions to enhance the gene transfection activity. The structure-activity relationship in this study is expected to offer a unique molecular design for drug/gene delivery systems.

Facile Method of Protein PEGylation by a Mono-Ion Complex.

Diethylaminoethyl end-modified poly(ethylene glycol) (DEAE-PEG) has been synthesized for the noncovalent PEGylation of proteins. The resulting DEAE-PEG and catalase formed an ion complex, that is, a protein mono-ion complex (MIC). The formation of the protein MIC was confirmed by native poly(acrylamide) gel electrophoresis and gel-filtration chromatography. The resulting catalase MIC preserved the catalase activity, confirmed by monitoring the O2 concentration with a Clark-type oxygen electrode, in spite of MIC formation. The catalase activity of the protein MIC was protected in the presence of a protease, trypsin, or 10% fetal bovine serum. Furthermore, less change in the circular dichroism measurements of the catalase MIC was observed as compared to those of a catalase-PEG conjugate (covalent PEGylation), suggesting less influence of the protein conformation. Consequently, the formation of the MIC is considered to be a facile method of protein PEGylation.

Lactoferrin-modified nanoparticles loaded with potent antioxidant Mn-porphyrins exhibit enhanced antioxidative activity in vitro intranasal brain delivery model.

In this study, for efficient intranasal brain delivery, we have prepared lactoferrin (Lf)-modified nanoparticles loaded with an amphiphilic Mn-porphyrin derivative, MndMImP3P (MnP) (Lf-NP-MnP). MndMImP3P was loaded into the nanoparticles with good loading efficiency. The stability of the resulting Lf-NP-MnP was sufficient for intranasal brain delivery. Lf on the surface of Lf-NP-MnP was significantly recognized by the Lf receptor, which leads to enhanced cellular uptake of MnP and efficient transcytosis. Furthermore, Lf-NP-MnP exhibited antioxidative activity in vitro experiment using a transwell assay as a model of intranasal brain delivery. Our result in this study is one step forward for efficient brain delivery of Mn-porphyrin derivatives to cure neurodegenerative diseases.

Fabrication and electrolyte characterization of uniaxially-aligned anion conductive polymer nanofibers.

We report the anion transport properties of anion conductive polymer nanofibers fabricated using an electrospinning method. The aligned nanofibers were prepared to evaluate the anion conductivity of the nanofibers. The aligned nanofibers had 10-15 times higher conductivity (up to 160 mS cm-1 at 90 °C and 95%RH) and lower activation energy (23-25 kJ mol-1) than the corresponding membranes, even though the nanofibers showed lower water uptake than the corresponding membranes. The anion conductivity measurement of nanofibers with different IEC values and anion species revealed that the dependency of anion conductivity on these factors was smaller in the nanofibers than in the corresponding membranes. These results indicate that effective ion transport pathways were formed in the nanofibers due to the phase separation and the polymer chain orientation along the nanofiber axis. These nanofibers are expected to be useful for future applications in alkaline fuel cells, air batteries, and other energy- and environment-related devices regardless of the ion species.

Screening for Methylated Poly(l-histidine) with Various Dimethylimidazolium/Methylimidazole/Imidazole Contents as DNA Carrier.

: Methylated poly(l-histidine) (PLH-Me), our original polypeptide, has controlled the contents of dimethylimidazolium, τ/π-methylimidazole and imidazole groups for efficient gene delivery. The screening for the PLH-Me as DNA carrier has been carried out by use of the PLH with 25 mol% (τ-methyl, 16 mol%; π-methyl, 17 mol%; deprotonated imidazole, 41 mol%), 68 mol% (τ-methyl, 16 mol%; π-methyl, 8 mol%; deprotonated imidazole, 8 mol%) and 87 mol% (τ-methyl, 7 mol%; π-methyl, 4 mol%; deprotonated imidazole, 2 mol%) dimethylimidazolium groups, that is, PLH-Me(25), PLH-Me(68) and PLH-Me(87), respectively. The screening of the chemical structure of PLH-Me has been carried out for DNA carrier properties, which are the stability of its DNA polyion complexes and gene expression. The DNA complexes with the 25 mol% and 68 mol% dimethylated PLH-Me possessed almost same ability to retain DNA, as compared with the 87 mol% dimethylated PLH-Me, which was examined by competitive exchange with dextran sulfate. From the gene transfection experiment against HepG2 cells, human hepatoma cell line, the PLH-Me(25)/DNA complex was revealed to mediate highest gene expression. These results suggest that the dimethyl-imidazolium/methylimidazole/imidazole balance of the PLH-Me is important for DNA carrier design.

Plasmid DNA mono-ion complex stabilized by hydrogen bond for in vivo diffusive gene delivery.

Our original concept of the mono-ion complex (MIC) between plasmid DNA (pDNA) and a monocationic biocompatible polymer has been stabilized by hydrogen bond formation. To form the hydrogen bond with pDNA, ω-amide-pentylimidazolium end-modified poly(ethylene glycol), that is, APe-Im-PEG, has been synthesized. Agarose gel retardation assay and circular dichroism measurement have revealed that the MIC between pDNA and APe-Im-PEG has been stabilized by the hydrogen bond between pDNA and the ω-amide group and that the stable MIC has surprisingly further migrated into gel, as compared with naked pDNA. The rise of melting temperature suggests that the specific hydrogen bond forms between an adenine-thymine base pair and the ω-amide group. The resulting pDNA MIC with APe-Im-PEG has enhanced gene expression by intramuscular administration in mice, as compared with a poly(ethylenimine) polyion complex (PIC). These results suggest that the pDNA MIC is diffusive in vivo administration site, as compared with pDNA PICs. Our methodology for MIC stabilization by a ω-amide group is expected to offer superior supramolecular systems to those by ubiquitous PICs for in vivo diffusive gene delivery.

A bioinspired polymer-bound Mn-porphyrin as an artificial active center of catalase.

The complex comprising a cationic Mn-porphyrin and carboxymethyl poly(1-vinylimidazole) (CM-PVIm) was prepared as an artificial active center of catalase. Interestingly, the catalase activity of the complex depends on the chain length of the polymer and the chemical structure of Mn-porphyrin. This study is one step forward in the development of a new class of water-soluble catalase mimics.

Dermal administration of manganese porphyrin by iontophoresis.

The present study describes a technique for dermal administration of cationic manganese porphyrin (Mn-porphyrin), an antioxidant with superoxide dismutase (SOD) activity, in hairless mouse. In general, the stratum corneum on the surface of the skin represents a barrier to passive diffusion of therapeutic agents by standard dermal administration. The present study investigated whether, dermal administration of Mn-porphyrin solution using iontophoresis, the electrical dermal administration technique, could overcome this barrier. We visually confirmed that Mn-porphyrin had penetrated to the reverse side of the hairless mouse skin after iontophoresis for a short period. With prolonged iontophoresis, the ratio of detectable Mn-porphyrin solution on the reverse side of the hairless mouse skin increased. In the future, this technique could provide an innovative approach for delivery of this antioxidant in intractable disease.

Synthesis of water-soluble dinuclear mn-porphyrin with multiple antioxidative activities.

Superoxide dismutase (SOD) and catalase activities of a drug are of great importance for its effective protection against reactive oxygen species (ROS)-induced injury. Achievement of catalase activity of a synthetic compound remains a challenge. Water-soluble Mn-porphyrins have high SOD and peroxynitrite (ONOO(-)) reducing activities, but not catalase-like activity. Herein, we are able to retain the fair SOD-like activity of a mononuclear Mn-5-(N-methylpyridinium-4-yl)-10,15,20-triphenyl porphyrin (MnM4PyP3P), while gaining in catalase-like activity with its dinuclear complex, 1,3-di[5-(N-methylene-pyridinium-4-yl)-10,15,20-triphenyl porphynato manganese] benzene tetrachloride (MnPD). Mechanistic study indicates that catalase-like activity of MnPD is due to synergism of two Mn active sites, where hydroxo-Mn(IV) complex is formed as an intermediate. The in vivo experiments demonstrate that MnPD significantly restores the treadmill-running ability of SOD-deficient mouse and thus indicates the therapeutic potential of MnPD. Furthermore, MnPD may serve as a mechanistic tool and indicate the new directions in the synthesis of catalase-like mimics.

Alkylimidazolium end-modified poly(ethylene glycol) to form the mono-ion complex with plasmid DNA for in vivo gene delivery.

In this study, we consider that the decrease in the transfection activity of polycations in vivo, compared with that in vitro, results from their polyion complex formation. Namely, owing to cross-linking between polycations and plasmid DNAs (pDNAs), the disadvantage of in vivo gene delivery mainly stems from the difficulty in controlling the properties of the resulting polyion complex at the nanoscale size. To avoid the cross-linking by polycations, we have establish the concept of "mono-ion complex" formation between pDNA and a monocationic biocompatible polymer. Here we have synthesized alkylimidazolium end-modified poly(ethylene glycol), that is, R-Im-PEG, and have tuned the electrostatic interaction between the resulting alkylimidazolium group and the phosphate group of pDNA by the length of the alkyl chain to achieve "mono-ion complex" formation with pDNA for in vivo gene delivery. Instead of a polyion complex, our original concept of the "mono-ion complex" consisting of the Bu-Im-PEG and pDNA is expected to offer unique tools to break through the barriers of in vivo gene delivery. As well as the field of gene delivery, this study is considered to have exploded the common sense that it is impossible to form not a polyion complex but a "mono-ion complex" under aqueous conditions for all fields of the modification of biomacromolecules.

Intracellular co-delivery of zinc ions and plasmid DNA for enhancing gene transfection activity.

Zinc ions, methylated poly(1-vinylimidazole) (PVIm-Me) and plasmid DNA (pDNA) have formed ternary complexes for gene delivery. The resulting Zn-PVIm-Me-pDNA complexes have delivered both Zn(2+) ions and pDNA inside cells, leading to the nuclear translocation of the pDNA. By use of the pDNA containing a nuclear protein, NF-κB, binding sequence, the intracellular co-delivery of Zn(2+) ions and pDNA has enhanced gene expression. These results suggest that the intracellular Zn(2+) ions delivered by Zn-PVIm-Me-pDNA complexes activated the NF-κB, enhancing the nuclear translocation of the pDNA. In conclusion, it has been demonstrated that the Zn-PVIm-Me-pDNA complex is capable of enhancing the gene transfection activity by a synergic effect of the PVIm-Me and the co-delivered intracellular Zn(2+) ions.

Superoxide dismutase activity of the naturally occurring human serum albumin-copper complex without hydroxyl radical formation.

The superoxide radical anion (O2(.-)) is biologically toxic and contributes to the pathogenesis of various diseases. Here we describe the superoxide dismutase (SOD) activity of human serum albumin (HSA) complexed with a single Cu(II) ion at the N-terminal end (HSA-Cu complex). The structure of this naturally occurring copper-coordinated blood serum protein has been characterized by several physicochemical measurements. The O2(.-) dismutation ability of the HSA-Cu (1:1) complex is almost the same as that of the well-known SOD mimics, such as Mn(III) -tetrakis(N-methylpyridinium)porphyrin. Interestingly, the HSA-Cu complex does not induce a subsequent Fenton reaction to produce the hydroxyl radical (OH(.)), which is one of the most harmful reactive oxygen species.

Nickel nanoparticle chains inside carbonized polymer nanofibers: preparation by electrospinning and ion-beam irradiation.

Novel organic-inorganic composite 1D nanostructures composed of carbonized conductive nanofibers and nickel nanoparticle chains were prepared by the combination of an electrospinning method from metal complex-containing polymer solutions and an ion-beam irradiation technique. The nickel nanoparticle chains were formed by self-assembly inside the carbonized nanofibers, which were characterized by STEM-HAADF observation and XPS spectroscopy. The existence of nickel complexes and the subsequent formation of nickel nanoparticles enhanced the electrical conductivities of carbonized nanofibers to reach above 0.5 S cm(-1) for the 6FDA-6FAP nanofibrous membrane containing 5.0 wt% Ni(acac)2 after ion-beam irradiation at an ion fluence of 1 × 10(16) ions per cm(2).